Abstract

Zymography is an electrophoretic technique enabling visualization of the number and approximate size of peptidases in a sample
on the basis of their hydrolysis of a protein substrate within the gel. The technique is particularly useful for analyzing
the peptidase composition of complex biological samples because visualization depends directly on proteolytic activity. This
unit presents a representative zymography protocol for the study of matrix metallopeptidases (MMPs).

Figure 21.15.3 A sample containing 100 pg recombinant proMMP‐2/MMP‐2 (rE) and 2 µl conditioned medium from TPA‐treated HT1080 (H) were run
on a 7.5% gelatin zymogram as in Figure . The effects of various proteinase inhibitors on activity were investigated. Compared with zymograms incubated in the absence
of inhibitors (none), addition of 20 µM E‐64, 10 µM pepstatin A (pepA), or 1 mM AEBSF to the renaturing and developing buffers
had no effect on enzyme activity. Addition of 20 mM EDTA, however, abolished all zones of gelatin digestion, confirming that
the enzymes are metalloproteinases. Lane M contains protein molecular‐mass markers.

Figure 21.15.4 MMP samples were analyzed on a 7.5% acrylamide gelatin zymogram, incubated in developing buffer overnight at 37°C. Lane 1
shows 100 pg MMP‐2 activated with 1 mM APMA, which results in a mixture of the 69‐kDa full‐length active form and the truncated
45‐kDa catalytic domain. Preincubation of this sample with an equimolar amount of TIMP‐2 for 1 hr at 37°C has no effect on
the observed pattern of digestion because the enzyme/inhibitor complex dissociates in sample loading buffer (lane 2). Addition
of a 50‐fold molar excess of α2M to MMP‐2 for 30 min at 37°C reduces the activity of both the 68‐ and 45‐kDa proteolytically active species (lane 3). α2M differs from TIMP‐2 in that the α2M/MMP‐2 complex is stable in sample loading buffer. Preincubation of MMP‐2 with TIMP‐2 protects it from association with α2M (lane 4), since the MMP‐2/TIMP‐2 complex is not proteolytically active and hence unable to bind to α2M. Lane 5 shows a mixture of 72‐kDa proMMP‐2, 68‐kDa MMP‐2, and 45‐kDA MMP‐2 catalytic domain, preincubated with TIMP‐2. Addition
of α2M to this sample has no effect on the zones of hydrolysis observed for proMMP‐2 as the zymogen is proteolytically inactive
(lane 6). The MMP‐2/TIMP‐2 complex is similarly inactive and does not interact with α2M (lane 6). The truncated catalytic domain, however, interacts poorly at TIMP‐2 and becomes associated with α2M (Itoh et al., ), shifting its zone of digestion from 45 kDa. A zone of lysis can often be observed associated with α2M. Lane M shows protein molecular mass markers.